The broad, long-term objectives of this proposal are: 1) To develop a complete molecular level view of soluble guanylate cyclase (sGC) activation and deactivation by nitric oxide (NO), and 2) To provide a rational basis for the understanding and treatment of human disease through the manipulation of NO. The specific aims are: 1) The characterization of NO activation and deactivation of sGC, including the nature of the second NO binding site, 2) The characterization of the nucleotide allosteric site, including the role of this site in modulating NO activation of the enzyme, 3) The mapping of the (a) intramolecular and (b) intermolecular protein-protein interactions that are important for sGC function, 4) To develop novel alkyl-NO ligands that are specific for the iron-heme binding site, to be used as spectroscopic probes, potential agonists and/or antagonists, and tools to help identify non-heme NO binding sites, and 5) obtain a crystal structure of sGC. The research design and methods for achieving these goals will be the use of basic molecular biology to acquire purified protein. Then, sGC function will be investigated with biochemical methods, including activity experiments and electronic absorption, resonance Raman, and electron paramagnetic spectroscopy. These studies are directed towards the development of a physiological model of sGC function. Understanding this function is critical for the design of therapeutic agents to treat diseases that involve the NO signaling pathway. Nitric oxide and nitric oxide synthase inhibitors are in clinical use in the treatment of diseases such as septic shock, pulmonary hypertension, male impotence, and angina pectortis. New therapies based on the manipulation of NO function in human biology will continue to have significant impact on human health and disease treatment/prevention. The proposed research examines the details of NO activation of sGC, the heart of this manipulation. [unreadable] [unreadable] [unreadable]